Background: Brucellosis is among the most widespread zoonotic infections estimated at 14% in Uganda. A cross-sectional study was conducted to estimate the sero-prevalence, risk factors of Brucella infection and malaria among abattoir workers. Methods: A survey was conducted among 232 abattoir workers in main abattoirs of Kampala and Mbarara districts in February 2007. A pre-tested questionnaire captured socio-demographic and occupational data. Brachial vein blood was tested for Brucella using Microplate Agglutination Test (MAT) and Standard Tube Agglutination Test (STAT) with a cut off titre of 1:160, and giemsa stained blood slides for malaria. Data was analyzed in SPSS 17.0. Results: Seven males (3%, n = 232) had malaria and dual brucella and Plasmodium falciparum malaria was found in one person. Brucella sero-positivity was 10% (95% CI 6 – 16; n = 232) with 12% (n = 161) in Kampala and 7% (n = 71) in Mbarara district. Non-use of protective gear Odds ratio (OR 3.3, 95% CI (1.25 – 50) and working in the abattoir beyond 5 years OR 2.4 95% CI (1.4 – 5.6) were associated with increased risk of Brucella infection. Age, sex, religion, keeping animals and consumption of raw milk or products were not significant. Conclusions: Brucella infection is a real risk among abattoir workers and use of full protective gear reduced risk significantly. Sensitization and public health care programs are needed to control this emerging problem.